PROJECT SUMMARY Hirschsprung?s disease (HSCR) is a birth defect caused by a number of gene mutations, resulting in distal colon that lacks an enteric nervous system (ENS). Children with HSCR are often treated by surgery to remove the distal bowel where the ENS is missing (called ?aganglionic bowel?), with the hope that the remaining ENS- innervated colon will exhibit normal function. However, after surgery up to 50% of children with HSCR have ongoing problems that are thought to be due to defects in the ENS-innervated colon. Numerous studies have identified disproportionate increases in the number of inhibitory enteric neurons in proximal, ganglion cell- containing colon tissue from HSCR patients and HSCR mouse models, raising the possibility that an imbalance in inhibitory/excitatory neurotransmission may be the underlying cause of continued bowel dysmotility after surgery. In order to improve colon dysmotility in HSCR, we first need a better understanding of the contributions of specific subtypes of myenteric neurons and ICC to colon motility patterns, and then identify which ENS/ICC circuits are altered in HSCR. Our lab has employed newly developed optogenetic tools combined with a technically innovative ex vivo preparation that keeps intrinsic and extrinsic ENS circuits intact, and we have generated preliminary data describing these circuits and the distinct patterns of contractility they produce in the adult mouse colon. For this proposal, I will use these techniques in two well-established mouse models of HSCR (piebald lethal and Ret+/-) to test the hypothesis that disruption in inhibitory and excitatory circuits in the proximal colon leads to abnormal colon motility behavior. In Aim 1, I will identify alterations in synaptic connectivity in intrinsic ENS/ICC circuits due to HSCR-associated mutations, reveal which subtypes of myenteric neurons are most affected, and directly correlate these changes to altered patterns of contractility. Aim 2 will determine whether extrinsic parasympathetic input to ENS/ICC circuits and resulting contractile responses are abnormal in HSCR mouse models. Finally, in Aim 3, I will optogenetically stimulate (using the light-activated ion channel, channelrhodopsin, ChR2) excitatory and inhibitory enteric neurons to determine the effects of manipulating excitatory/inhibitory tone on motility patterns in HSCR mouse models. Overall, the proposed experiments will identify specific ENS/ICC circuit defects that produce dysmotility in mouse models of HSCR and determine whether neuromodulation of the ENS-innervated colon, using external electrical or optogenetic stimulation, is capable of correcting circuit defects to normalize bowel function.